Linear quantitation of Aβ aggregation using Thioflavin T:: Reduction in fibril formation by colostrinin

Thioflavin T (ThT) fluorescence is a commonly used method to monitor A beta protein fibril formation. This method is particularly attractive since ThT fluoresces only when bound to fibrils, the reaction is completed within 1 min and ThT does not interfere with aggregation of A beta fibrils. One of the drawbacks of this method is the lack of a strict quantitative relationship between ThT fluorescence and fibril content. It was observed that, when the same gram molecular weight of A beta (1-40) is dissolved into varying amounts of base then placed into a constant volume of aqueous buffer, a non-linear fluorescent response is obtained. By maintaining a strict relationship between A beta content and the volume of base, this anomalous result can be alleviated and a linear dose response curve is obtained at much lower A beta concentrations than is typically observed. In addition, differences in A beta batch to batch preparations are alleviated. It was previously reported that colostrinin (CLN), a proline-rich peptide derived from colostrum, reduces fibril content and protects neuroblastoma cells against A beta peptide-induced toxicity. The newly developed ThT fluorescence protocol was used to quantify A beta fibril content after treatment with CLN. We also demonstrate that CLN, can solubilize A beta fibrils in a dose and time-dependent fashion. (c) 2006 Elsevier B.V. All rights reserved.